The overall objective of the research proposed here is to decipher the relationships between redox balance, oxidative stress, and metal ion metabolism in eukaryotic cells and their roles in human disease and aging. In particular, the detailed nature of superoxide stress, i.e., that component of oxidative stress which is due to direct chemical reactions of superoxide within living cells, will be sought. The principal model system is the budding yeast Saccharomyces cerevisiae, but hypotheses derived from the yeast studies will be tested in mice as well. The principal proteins to be studied are copper-zinc superoxide dismutase (CuZnSOD), which is an antioxidant enzyme, and its copper chaperone (CCS), which is believed to activate CuZnSOD by inserting copper. The first major goal is to clarify the role of iron in superoxide stress. It will be approached by studying the nature of the free iron that accumulates in organisms lacking CuZnSOD, the inactivation of certain iron-containing proteins by superoxide, and the mechanisms by which genetic suppressors decrease the superoxide sensitivity of yeast strains lacking CuZnSOD. The second major goal is to discover the relationship(s) between copper and zinc metabolism, superoxide stress, and the metallation state of CuZnSOD. Studies will be directed at understanding the mechanism of copper insertion into CuZnSOD by CCS and at determining the state of metallation of CuZnSOD in different tissues. Understanding how metallation of CuZnSOD is accomplished and how it is regulated in various tissues will help us better understand human antioxidant defenses and thus certain disease states. Overall, this work will advance our understanding of the detailed chemical mechanisms of superoxide stress, the roles of iron, copper, and zinc in the manifestation of superoxide stress, and, ultimately, the role of superoxide stress in human health and aging.